Filoviruses, e.g., ebolavirus and marburgvirus, cause severe hemorrhagic fevers in humans, with mortality rates reaching 88% (Feldmann, et al., 2003, Nat Rev Immunol, 3 (8):677-685) as well as epizootics in nonhuman primates and probably other mammals. Due to weaponization of marburgvirus by the USSR, the high fatality rates, and the potential for aerosol transmission filoviruses have been classified as Category A NIAID Priority Pathogens. There are currently no vaccines or therapeutics against filoviruses. The main filovirus species causing outbreaks in humans are ebolaviruses Zaire (EBOV) and Sudan (SUDV), as well as the Lake Victoria Marburg virus (MARV). Filoviruses are enveloped, single-stranded, negative sense RNA filamentous viruses and encode seven proteins, of which the spike glycoprotein (GP) is considered the main protective antigen. EBOV and MARV GP can be proteolytically cleaved by furin protease into two subunits linked by a disulfide linkage: GP1 (˜140 kDa) and GP2 (˜38 kDa) (Manicassamy, et al., 2005, J Virol, 79 (8):4793-4805). Three GP1-GP2 units form the trimeric GP envelope spike (˜550 kDa) on the viral surface (Feldmann, et al., 1993, Arch Virol Suppl, 7:81-100; Feldmann, et al., 1991, Virology, 182 (1):353-356; Geisbert and Jahrling, 1995, Virus Res, 39 (2-3):129-150; Kiley, et al., 1988a, J Gen Virol, 69 (Pt 8):1957-1967). GP1 mediates cellular attachment (Kiley, et al., 1988b, J Gen Virol, 69 (Pt 8):1957-1967; Kuhn, et al., 2006, J Biol Chem, 281 (23):15951-15958), and contains a mucin-like domain (MLD) which is heavily glycosylated and variable and has little or no predicted secondary structure (Sanchez, et al., 1998, J Virol, 72 (8):6442-6447).
It is well established that the filovirus GP represent the primary protective antigens (Feldmann, et al., 2003, Nat Rev Immunol, 3 (8):677-685; Feldmann, et al., 2005, Curr Opin Investig Drugs, 6 (8):823-830; Geisbert, et al., 2010, Rev Med Virol, 20(6):344-57). GP consists of a receptor binding GP1 subunit connected with the GP2 fusion domain via a disulfide link (FIG. 1). We have previously identified a specific region of the MARV and EBOV GP1 consisting of ˜150 amino acids (Kuhn, et al., 2006, J Biol Chem, 281 (23):15951-15958) that binds filovirus receptor-positive cells, but not receptor-negative cells, more efficiently than GP1, and compete with the entry of the respective viruses (Kuhn, et al., 2006, J Biol Chem, 281 (23):15951-15958). These properties are similar to regions defined for SARS coronavirus and Machupo arenavirus (Li, et al., 2003, Nature, 426 (6965):450-454; Radoshitzky, et al., 2007, Nature, 446 (7131):92-96; Wong, et al., 2004, J Biol Chem, 279 (5):3197-3201). This region of GP is referred to here as receptor binding region (RBR) and is part of a larger domain that excludes the variable, glycosylated, and bulky mucin-like domain (MLD). The RBR shows the highest level of homology between Filovirus glycoproteins (Kuhn, et al., 2006, J Biol Chem, 281 (23):15951-15958) as shown in FIG. 2. Therefore, the RBR represents a potential target for pan-filovirus antibodies.
The crystal structure of a trimeric, pre-fusion conformation of EBOV GP (lacking MLD) in complex with a EBOV-specific neutralizing antibody, KZ52 was solved at 3.4 Å (Lee, et al., 2008, Nature, 454 (7201):177-182). In this structure, three GP1 subunits assemble to form a chalice, cradled in a pedestal of the GP2 fusion subunits, while the MLD restricts access to the conserved RBR, sequestered in the GP chalice bowl. Ebola and Marburg GPs are cleaved by cathepsin proteases as a step in entry reducing GP1 to an ˜18 kDa product (Chandran, et al., 2005, Science, 308 (5728):1643-1645; Kaletsky, et al., 2007, J Virol, 81 (24):13378-13384; Schomberg, et al., 2006, J Virol, 80 (8):4174-4178). The structures suggest that the most likely site of cathepsin cleavage is the flexible β13-β14 loop of GP1 and illustrate how cleavage there can release the heavily glycosylated regions from GP, leaving just the core of GP1, encircled by GP2, with the RBR now well exposed. Cathepsin cleavage enhances attachment; presumably better exposing the RBR for interaction with cell surface factors trafficked with the virus into the endosome (Dube, et al., 2009, J Virol, 83:2883-2891).
Role of Antibodies in Protection Against Filovirus Hemorrhagic Fever.
While both T and B cell responses are reported to play a role in protective immune responses to filoviruses (Warfield, et al., 2005, J Immunol, 175 (2):1184-1191), a series of recent reports indicate that antibody alone can provide protection. Dye et al showed that purified convalescent IgG from macaques can protect non-human primates (NHPs) against challenge with MARV and EBOV when administered as late as 48 h post exposure (Dye, et al., 2012, Proc Natl Acad Sci US A, 109(13):5034-9). Olinger et al reported protection from EBOV challenge in NHPs treated with a cocktail of three monoclonal antibodies (mAbs) to GP administered 24 h and 48 h post exposure (Olinger, et al., 2012, Proc Natl Acad Sci USA, 109 (44):18030-18035). Similar results were also reported in two other studies (Qiu, et al., 2013, Sci Transl Med, 5 (207):207ra143; Qiu, et al., 2013, J Virol, 87 (13):7754-7757). Collectively these data demonstrate that a humoral response can control, alleviate, reduce, or prevent, filovirus infection.
To further explore the role of antibodies in protection against filoviruses in the context of vaccination, we performed an analysis of historical data from studies performed with virus-like particle (VLP) vaccines in >120 macaques to evaluate the relationship between protection from lethal challenge with antibody response to purified EBOV or MARV purified recombinant glycoproteins without the mucin-like domain and transmembrane region (GPddmuc). It was observed that an increase in antibody levels against the GPddmuc antigens can be associated with an increased probability of survival following lethal challenge (FIG. 3A). This relationship was not observed in the antibody levels to the matrix protein VP40 or irradiated, whole EBOV antigen (not shown). Analysis of the neutralizing antibody titer also demonstrated an association with survival for EBOV, supporting the hypothesis that neutralizing antibodies recognizing the RBR can provide protection from lethal infection. The majority of the data shown in FIG. 3A are from studies with VLPs expressing GP, VP40, and the nucleoprotein NP. Since it is known that NP induces strong cytotoxic T cell responses (Wilson and Hart, 2001, J Virol, 75 (6):2660-2664), it is possible that contribution of anti-NP T cell response to protection can impact our ability to fully decipher the role of antibodies in this analysis. Therefore, we analyzed data from a recent study using VLPs expressing GP and VP40. Fifteen cynomolgus macaques were vaccinated twice with various doses of GP/VP40 along with QS21 adjuvant and challenged 28 days later with 1000 PFU of EBOV. Both controls and nine of the vaccinated NHP died while six animals survived. Analysis of antibody response to GPddmuc in sera of these animals demonstrated a clear relationship between antibody titers to GPddmuc and survival with an apparent cut off at an antibody titer of ˜2000 AU/ml (FIG. 3B). This correlation became more obvious when the time of death of these animals was plotted against the antibody titer (FIG. 3B). One animal with an antibody titer below 2000 survived the challenge and this animal was very sick through day 14. This clearly indicates that vaccination with the GPddmuc proteins and likely proteins containing only the RBR could generate antibodies that could provide protection against infection.